CRISPR-Cas9 is the most commonly used gene editing tool in laboratories nowadays. This technique shows excellent simplicity and efficiency. Reversely, the flaw of it as the high potential rate of off-targeting is also non-negligible. Spacers are short sequence segments similar to phage or DNA, and it’s being used in the CRISPR-Cas9 technique to enable the insertion of an exogenous DNA sequence into part of the internal genes system. To decrease the off-targeting rate of CRISPR-Cas9 and enhance this gene editing tool into a more precise one, we chose the casposons as a gene editing tool and decided to change the spacer used in this technique into pUC19-TSD plasmids according to casposons. By this means, we would receive a gene editing tool that’s more reliable and safer to use (Figure 1).

In our project, we use casposons to achieve gene insertion. For easy detection, we chose the resistance gene, Kanamycin, as the target gene and set up a precise gene editing system in vitro.

In order to insert the TIR sequence into the pUC19 plasmid (Figure 2A), we use PCR to amplify the target DNA fragment and inserted the amplicons into DH10b competent cells. Inoculated a single colony into LB (Amp) culture medium, extracted the plasmid, and send to the company for Sanger sequencing (Figure 2B). As shown in the sequencing result, we successfully constructed the plasmid.

In order to obtain our target genes, we amplified different lengths of the target genes containing the Kanamycin gene fragment from the pUC19-DONER plasmid. In order to successfully amplify the genes, we use different annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 3).

In figure3, a clear and single DNA band at 1kp can be seen, indicating that we successfully amplified our target genes. We extracted the DNA fragments and stored them at -20℃ for future use.

To verify whether the long fragment gene with TIR sequence could be inserted into the TSD sequence effectively and correctly, the protein casposase was added for reaction, and the reaction products were recovered. Mixed components according to the table below, reacted in a metal bath at 37°C for 1h. Add PK enzyme at 37°C for 30min, then 95°C for 10min to terminate the reaction, added isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate containing both Kanamycin and Ampicillin antibiotics, incubated at 37℃ overnight. The next day, we calculated the number of colonies on the plate (Figure 4).

We inoculate the single colony in the LB liquid culture medium (Kana+Amp), extracted plasmids, amplified the target-gene-containing fragments, and send the company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 5), and the plasmids were successfully constructed. So far, we have successfully developed our gene editing system.

As the result shown above, compared with the negative control, we can find that with the casposase in the reaction system we successfully inserted the Kanamycin gene into the pUC19 plasmid so that the strain could grow on the plate containing antibiotics. So that we successfully developed a precise gene editing system in vitro, and this system could be used in future use or even be applied in clinical treatment.

We have already collected the figures from our experiments. Casposons is a new gene editing tools that can implement gene insertion precisely. In our project, we set up an in vitro gene editing system with the casposons. From the result, we can find out that with the casposase protein in this system, we inserted the Kanamycin gene into the pUC19 plasmid at the TSD site.
Because there are many diseases caused by gene variation, such as gene duplication, gene deletion, gene mutation, and so on, it is important to develop a gene editing tool to solve these problems at its source. Casposons is one of the tools nowadays. In the very near future, this tool will be improved and may be used in disease treatment and bring patients a better life.